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Jul 2012
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Quantification of Infectious Sendai Virus Using Plaque Assay
利用空斑实验量化分析传染性仙台病毒   

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Abstract

Sendai virus (SeV) is an enveloped, single-stranded RNA virus of the family Paramyxoviridae. SeV is a useful tool to study its infectious pathomechanism in immunology and the pathomechanism of a murine model of IgA nephropathy. Virus quantification is essential not only to determine the original viral titers for an appropriate application, but also to measure the viral titers in samples from the harvests from experiments. There are mainly a couple of units/titers for Sendai viral quantification: plaque-forming units (PFU) and hemagglutination (HA) titer. Of these, we here describe a protocol for Sendai virus plaque assay to provide PFU using LLC-MK2 cells (a rhesus monkey kidney cell lines) and Guinea pig red blood cells. This traditional protocol enables us to determine Sendai virus PFU in viral stock as well as samples from your experiments.

Keywords: Sendai virus (仙台病毒), Plaque assay (空斑实验), Titration (滴定法), Paramyxivirus (副粘病毒), Plaque-forming units (空斑形成单位), PFU (PFU)

Background

SeV is a mouse parainfluenza virus type I (Faisca and Desmecht, 2007) that was discovered in Sendai, Japan, in the 1950s (Ishida and Homma, 1978). SeV is a useful tool to study its infection and immune reaction (Fensterl et al., 2008; Chattopadhyay et al., 2010, 2011 and 2013; Yamashita et al., 2012a, 2012b and 2013; Veleeparambil et al., 2018) and the pathomechanism of a SeV-induced of IgA nephropathy (Yamashita et al., 2007; Chintalacharuvu et al., 2008). SeV is Precise viral quantification is essential to perform animal and cell culture experiments using an appropriate dose of SeV and also to obtain correct results from experimental samples containing SeV. In 1970s, SeV was quantitated by inoculation into embryonated eggs using hemagglutinin production as a criterion for infection (Shibuta et al., 1971). This method is highly sensitive but time consuming and complex. Therefore, a kidney cell-based plaque assay, a simple and reliable assay using hemadsorption (the attachment of red blood cells to the surface of cell monolayers infected with virus) has been developed (Jessen et al., 1987). This protocol provides a method for SeV PFU using LLC-MK2 cells (a rhesus monkey kidney cell lines) and Guinea pig red blood cells. This method can be applied for most types of samples including cell culture media, cell lysates, tissue homogenates, serum, urine, and bronchoalveolar lavage.

Materials and Reagents

  1. 96-well Polypropylene 1.2 ml Cluster Tubes (Sigma-Aldrich, catalog number: CLS4401-960EA)
  2. CorningTM 6-well plate (Thermo Fisher Scientific, catalog number: 07-200-83)
  3. CorningTM 10 ml pipettes (Thermo Fisher Scientific, catalog number: 07-200-574)
  4. 15 ml tubes (Thermo Fisher Scientific, catalog number: 12-565-268)
  5. Sendai virus (ATCC, catalog number: ATCC VR-105) as positive control
  6. HyClone® Characterized Fetal Bovine Serum, U.S. Origin (GE Healthcare Life Sciences, catalog number: SH30071.03HI)
  7. GibcoTM Gentamicin, 10 mg/ml (Thermo Fisher Scientific, catalog number: 11500506)
  8. L-Glutamine, 200 mM (Thermo Fisher Scientific, catalog number: A2916801)
  9. LLC-MK2 Original (ATCC, catalog number: CCL-7)
    Note: These cells are maintained in Medium 199 (Thermo Fisher Scientific, catalog number: 11150-067) containing 5% Fetal Bovine Serum, 20 μg/ml gentamicin, and 2 mM L-glutamine (complete Medium 199). LLC-MK2 cells should be used only up to about passage of 50. The plaques will become gradually smaller as the cell line ages.
  10. Guinea Pig Blood in Alsevers (Rockland antibodies & assays, catalog number: R305-0050)
    Note: This needs to be less than 2 weeks old.
  11. Medium 199 (10x) (Thermo Fisher Scientific, catalog number: 11825015)
  12. Medium 199 (1x) (Thermo Fisher Scientific, catalog number: 11150067)
  13. HBSS, calcium, magnesium (Thermo Fisher Scientific, catalog number: 14025092)
  14. Sodium Bicarbonate 7.5% solution (Thermo Fisher Scientific, catalog number: 25080094)
  15. BD Difco Agar (Fisher Scientific, catalog number: DF0812-17-9)
  16. Trypsin (0.25%), phenol red (Thermo Fisher Scientific, catalog number: 15050065)
    Note: The final concentration is 0.00025% (2.5 μg/ml). This reagent needs optimization for a particular lot. 2.5 μg/ml ± 0.25 μg/ml can make the difference between nice large plaques and the cells detached from the plates.
  17. Sterile PBS (with Ca2+ and Mg2+)
  18. Penicillin-Streptomycin (5,000 U/ml) (Thermo Fisher Scientific, catalog number: 15070063)
  19. Complete 2x medium (see Recipes)

Equipment

  1. Multichannel pipette (Gilson, catalog number: FA10015)
  2. Pipettes, P20, P200, P1000 (Gilson, catalog number: F167300)
  3. Biosafety cabinet (Thermo Fisher Scientific, catalog number: 1305)
  4. Tissue culture incubator (at 37 °C with 5% CO2) (Thermo Fisher Scientific, catalog number: 51025983)
  5. 100 ml glass bottles (Research Products International, catalog number: 219510)
  6. PrecisionTM General Purpose Water bath (Thermo Fisher Scientific, catalog number: TSGP02)
  7. Autoclave (Steris Amsco Eagle, catalog number: 3021-C Gravity Steam Sterilizer)
  8. Sonic water bath (Skymen Cleaning Equipment, model: JP-008)
  9. Vortex mixer (Research Products International, catalog number: 155560)
  10. Portable Mini Light Box, Benchtop Light Source (Research Products International, catalog number: 815500)

Procedure

Days -5 to -2
Seed LLC-MK2 cells into the necessary number of 6-well plates (as below) to test samples and controls at all needed dilutions.

Note: You want the plates with lightly confluent cells on Day 0 (Figure 1).


Figure 1. Lightly confluent LLC-MK2 cells. Once overlaid agar is solidified, cells can no longer proliferate. Therefore, the cells want to be confluent when you overlay agar on the cells (Scale bar = 100 μm).

Day 0

  1. Set up sterile 50 ml agar solution in 100 ml glass bottle(s) to be used in Step 3f
    1. Add 0.5 g agar to 50 ml distilled water in a 100 ml bottle. One to 4 bottles are needed.
    2. Autoclave the bottle(s) at 121 °C for 30 min.
    3. Mix the agar well when it comes out of the autoclave and hold in a 50 °C water bath until ready to use (Step 3f).
    Note: The final 100 ml will handle 8 six-well plates most comfortably at the same time. Up to total 4 bottles and 32 plates are practical.
  2. Viral preparation
    1. Thaw the virus samples and a positive control (to be titrated) on ice, and keep them on ice.
      Note: For the proper analysis, a positive control (Sendai virus, VR-105 from ATCC) is required for every experiment. One vial (1 ml) from ATCC can be used for 10 to 20 experiments. The vial from ATCC should be aliquoted into 50 μl to 100 μl each (10 to 20 tubes) in the first experiment, and stored in liquid nitrogen tank. The viral titer is stable at least for three years by this method according to our experience. We recommend you use the original SeV from ATCC (not a "homemade" SeV stock) as a positive control because of the certainty of the viral titer.
    2. Sonicate (40 kHz frequency and 35 W ultrasonic power) the virus samples for 30 sec in a sonic water bath at room temperature before use to break and dissociate viral chunks.
    3. Make serial dilutions of virus in pure Medium 199 (without Fetal Bovine Serum, gentamicin or L-glutamine) at whole log (10-fold) dilutions or half-log (3.16-fold) dilutions in the appropriate range (Figure 2). Doing these dilutions in rack of 96 microtubes (1.2 ml volume) with a multichannel pipette will greatly increase efficiency. Keep samples and dilutions on ice.
      Note: "Pure Medium 199" means only Medium 199 (Thermo Fisher Scientific) without serum.


      Figure 2. Serial dilutions of viral sample. A. For whole-log dilutions, 100 μl of sample will be serially transferred to the next tube that contains 900 μl of pure Medium 199. B. For half-log dilutions, 316 μl of sample will be serially transferred to the next tube that contains 684 μl of pure Medium 199.

  3. Inoculation and incubation
    1. Rinse lightly the confluent monolayers of LLC-MK2 cells on the plates with HBSS with Ca2+Mg2+: Remove old media and add 2 ml of HBSS with Ca2+Mg2+.
    2. Suction the media off the number of plates, and quickly add 200 μl of pure Medium 199 to each well.
    3. Add 200 μl of sample dilutions in duplicate or triplicate. Work from the most dilute to the least dilute to save on tip changes.
    4. Incubate plates for 90 min at 37 °C with 5% CO2.
      Note: Gently agitate plates about every 30 min to avoid drying out the plates.
    5. During the incubation, make up complete 2x media in 50 ml aliquots (see Recipes).
    6. At the end of the 90-min incubation, mix together the complete 2x media (at room temperature) and the 1% agar water (in a 50 °C water bath) for the first set of 8 plates, and place the mixture at room temperature.
    7. Suction viral inoculum media from the plates, and quickly add 2 ml of media with 0.5% agar (Step 3f) into each well using a 10 ml pipette (capacity of 12 ml).
    8. Cool at room temperature until the agar media solidify. Do not stack the plates.
    9. Incubate the plates (cells overlaid with the agar media) for 3 days at 37 °C with 5% CO2.

Day 3: Hemadsorption
  1. Wash approximately 3 ml of guinea pig blood with sterile PBS (with Ca2+ and Mg2+) 3 times in a 15 ml tube. Spin at 434 x g for 5 min to obtain about 1 ml pellet of red blood cells (RBCs).
  2. Resuspend the 1 ml of RBC pellet to 9 ml of PBS (10% volume/volume) with PBS (with Ca2+ and Mg2+).
  3. Make a 0.1% RBC suspension with PBS (with Ca2+ and Mg2+) from 10% RBC suspension.
  4. Remove 6-well plates from the incubator in small groups (like 4 plates).
  5. Shake the agar off one plate at a time with a quick snap of the wrist into a biohazard bag and immediately add 2 ml of PBS (with Ca2+ and Mg2+; at room temperature).
    Note: Removal of agar leaves the plate very dry and the cells are vulnerable (LLC-MK2 cells are not particularly sensitive to detachment, but this is an effect of this assay). Please add PBS immediately but gently along the wall of the wells, not directly to the cells.
  6. Remove PBS from the 6-well plates, and add 2 ml of 0.1% RBC solution to each well (Step 3).
  7. Incubate plates at room temperature for 20-30 min.
  8. Gently agitate plates to resuspend RBCs, and replaced the RBC suspension with 2 ml PBS (with Ca2+ and Mg2+).
    Note: This one-time wash is usually sufficient, but a second wash is acceptable.
  9. Score plaques as well-defined areas where RBCs attach (Figure 3).
    Note: Clean the bottom (outside) of the well with alcohol before counting. If you place the plates on a light box, you will be able to more easily identify the plaques.
  10. Calculate pfu/ml based on sample size (200 μl) and dilution.


    Figure 3. Sendai virus plaques. A. Five plaques in one well. B. Magnification of one plaque shows a ring-shape plaque of red blood cells attaching to LLC-MK2 cells and a central empty area where LLC-MK2 cells died and detached.

    An example of titer calculation
    If the inoculation sample in Figure 3 is 107 dilution:
    Virus titer (pfu/ml) = 5 (plaques)/[0.2 ml (inoculation volume) x 107 (sample dilution)] = 2.5 x 108 PFU/ml

Notes

This protocol needs an appropriate facility (Biosafety level 2) and an approved Institutional Biosafety Committee (IBC) protocol.

Recipes

  1. Complete 2x medium
    Note: All components need to be sterile before mixing.

Acknowledgments

Dr. Yamashita is supported by American Heart Association Scientist Development Grant (17SDG33660947) and University of California Los Angeles Clinical and Translational Science Institute Grant (ΜL1TR001881). We would like to thank Dr. Steven Emancipator for the mentoring in the early stage of Dr. Yamashita's career development and Dr. John Nedrud for previous collaboration.

Competing interests

The authors do not have any potential conflicts of interest or competing interests to declare.

References

  1. Chattopadhyay, S., Fensterl, V., Zhang, Y., Veleeparambil, M., Yamashita, M. and Sen, G. C. (2013). Role of interferon regulatory factor 3-mediated apoptosis in the establishment and maintenance of persistent infection by Sendai virus. J Virol 87(1): 16-24.
  2. Chattopadhyay, S., Marques, J. T., Yamashita, M., Peters, K. L., Smith, K., Desai, A., Williams, B. R. and Sen, G. C. (2010). Viral apoptosis is induced by IRF-3-mediated activation of Bax. EMBO J 29(10): 1762-1773.
  3. Chattopadhyay, S., Yamashita, M., Zhang, Y. and Sen, G. C. (2011). The IRF-3/Bax-mediated apoptotic pathway, activated by viral cytoplasmic RNA and DNA, inhibits virus replication. J Virol 85(8): 3708-3716.
  4. Chintalacharuvu, S. R., Yamashita, M., Bagheri, N., Blanchard, T. G., Nedrud, J. G., Lamm, M. E., Tomino, Y. and Emancipator, S. N. (2008). T cell cytokine polarity as a determinant of immunoglobulin A (IgA) glycosylation and the severity of experimental IgA nephropathy. Clin Exp Immunol 153(3): 456-462.
  5. Faisca, P. and Desmecht, D. (2007). Sendai virus, the mouse parainfluenza type 1: a longstanding pathogen that remains up-to-date. Res Vet Sci 82(1): 115-125.
  6. Fensterl, V., White, C. L., Yamashita, M. and Sen, G. C. (2008). Novel characteristics of the function and induction of murine p56 family proteins. J Virol 82(22): 11045-11053.
  7. Ishida, N. and Homma, M. (1978). Sendai virus. Adv Virus Res 23: 349-383. 
  8. Jessen, R. H., Nedrud, J. G. and Emancipator, S. N. (1987). A mouse model of IgA nephropathy induced by Sendai virus. Adv Exp Med Biol 216B: 1609-1618.
  9. Shibuta, H., Akami, M. and Matumoto, M. (1971). Plaque formation by sendai virus of parainfluenza virus group, type 1 on monkey, calf kidney and chick embryo cell monolayers. Jpn J Microbiol 15(2): 175-183. 
  10. Veleeparambil, M., Poddar, D., Abdulkhalek, S., Kessler, P. M., Yamashita, M., Chattopadhyay, S. and Sen, G. C. (2018). Constitutively bound EGFR-mediated tyrosine phosphorylation of TLR9 is required for its ability to signal. J Immunol 200(8): 2809-2818.
  11. Yamashita, M., Chattopadhyay, S., Fensterl, V., Saikia, P., Wetzel, J. L. and Sen, G. C. (2012a). Epidermal growth factor receptor is essential for Toll-like receptor 3 signaling. Sci Signal 5(233): ra50.
  12. Yamashita, M., Chattopadhyay, S., Fensterl, V., Zhang, Y. and Sen, G. C. (2012b). A TRIF-independent branch of TLR3 signaling. J Immunol 188(6): 2825-2833.
  13. Yamashita, M., Chintalacharuvu, S. R., Kobayashi, N., Nedrud, J. G., Lamm, M. E., Tomino, Y. and Emancipator, S. N. (2007). Analysis of innate immune responses in a model of IgA nephropathy induced by Sendai virus. Contrib Nephrol 157: 159-163.
  14. Yamashita, M., Millward, C. A., Inoshita, H., Saikia, P., Chattopadhyay, S., Sen, G. C. and Emancipator, S. N. (2013). Antiviral innate immunity disturbs podocyte cell function. J Innate Immun 5(3): 231-241.

简介

仙台病毒(SeV)是包虫病毒科(Paramyxoviridae)家族的包膜单链RNA病毒。 SeV是研究其在免疫学中的传染性病理机制和IgA肾病小鼠模型的病理机制的有用工具。 病毒定量不仅对确定适当应用的原始病毒滴度至关重要,而且对于测量来自实验收获的样品中的病毒滴度也是必不可少的。 仙台病毒量化主要有几个单位/滴度:噬斑形成单位(PFU)和血细胞凝集(HA)滴度。 其中,我们在此描述了使用LLC-MK2细胞(恒河猴肾细胞系)和豚鼠红细胞提供PFU的仙台病毒噬斑测定方案。 这种传统方案使我们能够确定病毒原种中的仙台病毒PFU以及实验样品。

【背景】 SeV是一种小鼠副流感病毒I型(Faisca和Desmecht,2007),它是在20世纪50年代在日本仙台发现的(Ishida和Homma,1978)。 SeV是研究其感染和免疫反应的有用工具(Fensterl et al。,2008; Chattopadhyay et al。,2010,2011 and 2013; Yamashita et al。,2012a,2012b和2013; Veleeparambil et al。,2018)和SeV诱导的IgA肾病的病理机制(Yamashita et al。 ,2007; Chintalacharuvu et al。,2008)。 SeV是精确的病毒定量对于使用适当剂量的SeV进行动物和细胞培养实验以及从含有SeV的实验样品获得正确结果是必需的。在20世纪70年代,通过使用血凝素产生作为感染标准接种到胚胎卵中来定量SeV(Shibuta 等,,1971)。该方法高度灵敏,但耗时且复杂。因此,基于肾细胞的噬斑测定,使用血细胞吸附(红细胞附着到感染病毒的细胞单层表面)的简单可靠的测定已被开发出来(Jessen et al。, 1987)。该方案提供了使用LLC-MK2细胞(恒河猴肾细胞系)和豚鼠红细胞的SeV PFU的方法。该方法可应用于大多数类型的样品,包括细胞培养基,细胞裂解物,组织匀浆,血清,尿液和支气管肺泡灌洗液。

关键字:仙台病毒, 空斑实验, 滴定法, 副粘病毒, 空斑形成单位, PFU

材料和试剂

  1. 96孔聚丙烯1.2毫升簇管(Sigma-Aldrich,目录号:CLS4401-960EA)
  2. Corning TM 6孔板(Thermo Fisher Scientific,目录号:07-200-83)
  3. Corning TM 10 ml移液器(Thermo Fisher Scientific,目录号:07-200-574)
  4. 15毫升管(Thermo Fisher Scientific,目录号:12-565-268)
  5. 仙台病毒(ATCC,目录号:ATCC VR-105)作为阳性对照
  6. HyClone ®表征胎牛血清,美国原产地(GE Healthcare Life Sciences,目录号:SH30071.03HI)
  7. Gibco TM 庆大霉素,10 mg / ml(Thermo Fisher Scientific,目录号:11500506)
  8. L-谷氨酰胺,200 mM(Thermo Fisher Scientific,目录号:A2916801)
  9. LLC-MK2原件(ATCC,目录号:CCL-7)
    注意:将这些细胞维持在含有5%胎牛血清,20μg/ ml庆大霉素和2mM L-谷氨酰胺(完全培养基199)的培养基199(Thermo Fisher Scientific,目录号:11150-067)中。 LLC-MK2细胞应仅使用至50左右。随着细胞系的老化,斑块将逐渐变小。
  10. Alsevers中的豚鼠血(Rockland抗体和检测,目录号:R305-0050)
    注意:这需要不到2周的时间。
  11. 中等199(10x)(Thermo Fisher Scientific,目录号:11825015)
  12. 中等199(1x)(赛默飞世尔科技,目录号:11150067)
  13. HBSS,钙,镁(赛默飞世尔科技,目录号:14025092)
  14. 碳酸氢钠7.5%溶液(Thermo Fisher Scientific,目录号:25080094)
  15. BD Difco Agar(Fisher Scientific,目录号:DF0812-17-9)
  16. 胰蛋白酶(0.25%),酚红(Thermo Fisher Scientific,目录号:15050065)
    注意:最终浓度为0.00025%(2.5μg/ ml)。该试剂需要针对特定批次进行优化。 2.5μg/ ml±0.25μg/ ml可以区分漂亮的大斑块和从平板上脱落的细胞。
  17. 无菌PBS(Ca 2 + 和Mg 2 + )
  18. 青霉素 - 链霉素(5,000 U / ml)(Thermo Fisher Scientific,目录号:15070063)
  19. 完成2x中等(见食谱)

设备

  1. 多通道移液器(Gilson,目录号:FA10015)
  2. 移液器,P20,P200,P1000(Gilson,目录号:F167300)
  3. 生物安全柜(Thermo Fisher Scientific,目录号:1305)
  4. 组织培养箱(37°C,5%CO 2 )(Thermo Fisher Scientific,目录号:51025983)
  5. 100毫升玻璃瓶(Research Products International,目录号:219510)
  6. 精密 TM 通用水浴(Thermo Fisher Scientific,目录号:TSGP02)
  7. 高压灭菌器(Steris Amsco Eagle,目录号:3021-C重力蒸汽灭菌器)
  8. 声波水浴(Skymen清洁设备,型号:JP-008)
  9. 涡旋混合器(国际研究产品,产品目录号:155560)
  10. 便携式迷你灯箱,台式光源(国际研究产品,产品目录号:815500)

程序

天-5到-2
将LLC-MK2细胞种入必要数量的6孔板(如下所示),以便在所有需要的稀释度下测试样品和对照。

注意:您希望第0天(图1)的平板具有轻微融合的细胞。


图1.轻度融合的LLC-MK2细胞。一旦覆盖的琼脂凝固,细胞就不能再增殖。因此,当您在细胞上叠加琼脂时,细胞想要融合(比例尺=100μm)。

第0天

  1. 在100ml玻璃瓶中放置无菌50ml琼脂溶液,用于步骤3f
    1. 在100ml瓶中加入0.5g琼脂至50ml蒸馏水中。需要一到四瓶。
    2. 将瓶子在121℃下高压灭菌30分钟。
    3. 将琼脂从高压灭菌器中取出时将其混合均匀,并保持在50℃水浴中直至准备使用(步骤3f)。
    注意:最终的100毫升将同时最舒适地处理8个六孔板。最多可容纳4瓶和32块板。
  2. 病毒制剂
    1. 在冰上解冻病毒样本和阳性对照(待滴定),并将它们保存在冰上。
      注意:为了进行适当的分析,每个实验都需要阳性对照(仙台病毒,来自ATCC的VR-105)。来自ATCC的一个小瓶(1ml)可用于10至20次实验。来自ATCC的小瓶应在第一个实验中等分为50μl至100μl(10至20个管),并储存在液氮罐中。根据我们的经验,通过该方法,病毒滴度至少稳定三年。我们建议您使用来自ATCC的原始SeV(不是“自制的”SeV原液)作为阳性对照,因为病毒滴度的确定性。
    2. 在室温下在声波水浴中超声处理(40kHz频率和35W超声功率)病毒样品30秒,然后用于破坏和分离病毒块。
    3. 在完全对数(10倍)稀释或半对数(3.16倍)稀释的适当范围内,在纯培养基199(不含胎牛血清,庆大霉素或L-谷氨酰胺)中连续稀释病毒(图2)。使用多通道移液器在96微管(1.2 ml体积)的架子中进行这些稀释将大大提高效率。将样品和稀释液保存在冰上。
      注意:“Pure Medium 199”仅指不含血清的Medium 199(Thermo Fisher Scientific)。


      图2.病毒样品的系列稀释液。 :一种。对于全对数稀释,将100μl样品连续转移至含有900μl纯培养基199的下一个管中.B。对于半对数稀释,将316μl样品连续转移至含有684的下一个管中。 μl纯培养基199。

  3. 接种和孵化
    1. 用HBSS和Ca 2 + Mg 2 + 轻轻冲洗板上LLC-MK2细胞的汇合单层:去除旧培养基并加入2 ml含Ca 2 + 镁 2 + 。
    2. 从多个平板吸出培养基,并快速向每个孔中加入200μl纯培养基199。
    3. 一式两份或一式三份加入200μl样品稀释液。从最稀释到最小稀释工作以节省尖端变化。
    4. 将板在37℃下用5%CO 2 孵育90分钟。
      注意:每30分钟轻轻搅拌一次,以避免干燥板。
    5. 在孵育期间,以50ml等分试样制备完整的2x培养基(参见食谱)。
    6. 在90分钟孵育结束时,将完整的2x培养基(在室温下)和1%琼脂水(在50°C水浴中)混合,用于第一组8个培养板,并将混合物置于室内温度。
    7. 从平板吸入病毒接种培养基,并使用10ml移液管(容量12ml)快速将2ml含有0.5%琼脂的培养基(步骤3f)加入每个孔中。
    8. 在室温下冷却直至琼脂培养基固化。不要堆叠板。
    9. 将平板(用琼脂培养基覆盖的细胞)在37℃,5%CO2下孵育3天。

第3天: Hemadsorption
  1. 在15ml管中用无菌PBS(含有Ca 2 + 和Mg 2 + )洗涤约3ml豚鼠血液3次。在434 x g 旋转5分钟以获得约1ml的红细胞(RBC)沉淀。
  2. 将1ml RBC沉淀重悬于含有PBS的9ml PBS(10%体积/体积)中(Ca 2 + 和Mg 2 + )。
  3. 从10%RBC悬浮液中用PBS(具有Ca 2 + 和Mg 2 + )制备0.1%RBC悬浮液。
  4. 从培养箱中取出6孔板,分成小组(如4块板)。
  5. 将手腕快速拍打到生物危害袋中,同时将琼脂从一个平板上摇下,然后立即加入2毫升PBS(C a 2 < / sup> + 和Mg 2 + ;室温下)。
    注意:去除琼脂会使平板非常干燥并且细胞容易受损(LLC-MK2细胞对脱离不是特别敏感,但这是该测定的效果)。请立即轻轻地沿着孔壁添加PBS,而不是直接添加到细胞中。
  6. 从6孔板中取出PBS,并向每个孔中加入2ml 0.1%RBC溶液(步骤3)。
  7. 在室温下孵育平板20-30分钟。
  8. 轻轻搅动平板重悬RBC,并用2 ml PBS替换RBC悬浮液(Ca 2 + 和Mg 2 + )。
    注意:这种一次性洗涤通常就足够了,但第二次洗涤是可以接受的。
  9. 将斑块评为红细胞附着的明确区域(图3)。
    注意:在计数前用酒精清洁井底(外部)。如果将平板放在灯箱上,您将能够更容易地识别斑块。
  10. 根据样本量(200μl)和稀释度计算pfu / ml。


    图3.仙台病毒斑块。 A.一个孔中有五个斑块。 B.一块牙菌斑放大显示附着在LLC-MK2细胞上的红细胞环形斑块和LLC-MK2细胞死亡和脱落的中央空区。

    滴度计算的一个例子
    如果图3中的接种样品是10 7 稀释度:
    病毒滴度(pfu / ml)= 5(噬菌斑)/ [0.2ml(接种体积)×10 7 (样品稀释)] = 2.5×10 8 PFU / ml

笔记

该协议需要适当的设施(生物安全级别2)和经批准的机构生物安全委员会(IBC)协议。

食谱

  1. 完成2x中等
    注意:所有组件在混合前都需要无菌。

致谢

Yamashita博士得到美国心脏协会科学家发展基金会(17SDG33660947)和加州大学洛杉矶分校临床和转化科学研究所基金(ΜL1TR001881)的支持。我们要感谢Steven Emancipator博士在Yamashita博士职业发展早期的指导和John Nedrud博士之前的合作。

利益争夺

作者没有任何潜在的利益冲突或竞争利益申报。

参考

  1. Chattopadhyay,S.,Fensterl,V.,Zhang,Y.,Veleeparambil,M.,Yamashita,M。和Sen,G。C.(2013)。 干扰素调节因子3介导的细胞凋亡在仙台病毒持续感染的建立和维持中的作用。 J Virol 87(1):16-24。
  2. Chattopadhyay,S.,Marques,J.T.,Yamashita,M.,Peters,K.L.,Smith,K.,Desai,A.,Williams,B.R。和Sen,G.C。(2010)。 通过IRF-3介导的Bax激活诱导病毒性凋亡。 EMBO J 29(10):1762-1773。
  3. Chattopadhyay,S.,Yamashita,M.,Zhang,Y。和Sen,G。C.(2011)。 IRF-3 / Bax介导的细胞凋亡途径,由病毒细胞质RNA和DNA激活,抑制病毒复制。 J Virol 85(8):3708-3716。
  4. Chintalacharuvu,S.R.,Yamashita,M.,Bagheri,N.,Blanchard,T.G。,Nedrud,J.G.,Lamm,M.E.,Tomino,Y。和Emancipator,S.N。(2008)。 T细胞因子极性作为免疫球蛋白A(IgA)糖基化的决定因素和实验性IgA肾病的严重程度。 Clin Exp Immunol 153(3):456-462。
  5. Faisca,P。和Desmecht,D。(2007)。 仙台病毒,小鼠副流感病毒1型:一种长期存在的病原体,一直保持最新状态。< / a> Res Vet Sci 82(1):115-125。
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  10. Veleeparambil,M.,Poddar,D.,Abdulkhalek,S.,Kessler,P.M.,Yamashita,M.,Chattopadhyay,S。和Sen,G.C。(2018)。 TLR9的组成型结合EGFR介导的酪氨酸磷酸化是其发出信号的能力所必需的。 J Immunol 200(8):2809-2818。
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引用:Tatsumoto, N., Miyauchi, T., Arditi, M. and Yamashita, M. (2018). Quantification of Infectious Sendai Virus Using Plaque Assay. Bio-protocol 8(21): e3068. DOI: 10.21769/BioProtoc.3068.
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